The thymus is a heterogeneous mix of hematopoietic and stromal cells that function to generate a functional, self-tolerant T-cell pool. Although many of these populations are well studied, the role of non-epithelial stroma remains unclear. Thymic mesenchyme has been identified as an important regulator of T-cell egress. Studies of lymphotoxin beta-receptor LTβR have revealed its critical role in T-cell egress as well as the development and function of lymph node mesenchyme. We hypothesized that LTβR regulation of thymic mesenchyme is critical forT-cell egress. To test this we generated \(Wnt-1^{cre}Ltbr^{flox}\) mice to delete LTβR on thymic mesenchyme and revealed this to be non-essential forT-cell egress. Moreover, we generated \(Foxn-1^{cre}Ltbr^{flox}\) mice to delete LTβR on thymic epithelial cell (TEC). Despite the critical role of LTβR in medullary TEC development, T-cell egress was normal. However, deleting LTβR on thymic endothelium using \(Flk-1^{cre}Ltbr^{flox}\) mice revealed an essential role of LTβR regulation of endothelium to control T-cell egress. Our analysis also revealed that T-cell entry into the perivascular space during T-cell egress occurs stochastically. Collectively our findings highlight a novel role for LTβR regulation of thymic endothelium as a critical pathway of T-cell egress.

Hepatitis C Virus (HCV) is a clinically important infection that leads to chronic liver disease and Human Immunodeficiency Virus (HIV) co-infected patients have more rapid progression to severe liver disease and show higher rates of HCV vertical transmission. Hepatocytes are a highly differentiated cell type and support low level HCV replication. Most studies of the viral life cycle use de-differentiated hepatoma cell lines, which are highly permissive. The mechanism behind this difference is poorly understood. We show that dimethylsulfoxide (DMSO) differentiated Huh-7 cells have a 100-fold reduction in permissivity to HCV infection. We confirm that these cells are differentiated and upregulate key liver specific markers including miR122. They are metabolically active and have intact innate signaling pathways in response to infection. We observed a 10-fold reduction in the initiation of replication and a 10-fold loss in extra-cellular particle infectivity. In contrast cell-to-cell dissemination rates were comparable and cell-contact dependent infection of differentiated cells can overcome the restrictions seen in cell-free infection. HCV cell-to-cell transmission can also be mediated by other cell types. T cells are the primary cell supporting HIV-1 infection. We have shown that HCV can bind primary and immortalized T cells and trans-infect hepatoma cells. This requires replicating HIV but is independent of co-receptor engagement. HIV-1 infection of CD4+ T cells induces a significant increase in HCV trans-infection by increased viral binding. T cells provide a vehicle for HIV-1 to promote HCV infectivity, transmission and persistence.

Kaposi’s sarcoma-associated herpes virus (KSHV) encodes genes with immunomodulatory potential, one of which is vIRF-2 that shares homology to cellular interferon regulatory factors. The innate antiviral mechanism mediating the type I interferons is an essential host cell defence mechanism limiting viral replication. The aim of this study was to determine the range and type of cellular gene sets and associated biological pathways whose expression is deregulated by vIRF-2. HEK 293-derived cell clones were engineered to express doxycycline-inducible vIRF-2. Interferon (IFN) responses were induced with recombinant (r) IFN-α and measured by an IFN stimulated response elements (ISRE) luciferase reporter gene assay. The effects of vIRF-2 on cell transcriptome profile in response to rIFN-α were determined by DNA microarray analysis and confirmed by immunoblot assay. vIRF-2 protein inhibited activation of ISRE-luc by over 50% and significantly (p<0.05) down-regulated the expression of 57/78 (73%) of rIFN-α regulated genes. The DAVID and GSEA software packages revealed vIRF-2 down-regulates the RIG-I-like receptor, JAK-STAT and Ubiquitin ligase pathways and many gene sets involved in antiviral response, transcriptional regulation and apoptosis. Immunoblot assays demonstrated reduced levels of RIG-I/DDX58, TBK-1, p-38, STAT1, pSTAT1, IRF-9 and OAS3. The biological significance of the vIRF-2 anti-IFN property was demonstrated by the rescue of encephalomyocarditis virus (EMCV) replication in vIRF-2 expressing cells treated with rIFN-α; EMCV was titred by plaque assay on L929 cells. These data confirm the role of KSHV vIRF-2 in negative regulation of the IFN-α/β innate immune response by a mechanism dependent on negative regulation of RIG-I/DDX58, STAT1, IRF-9 and OAS3.

Arboviruses (Arthropod-borne viruses) cause acute diseases that are increasingly affecting both human and animal health. Currently, there is a critical lack of understanding about the nature of arbovirus-host interactions in the lymph nodes (LNs), the place where the adaptive immune response is initiated and shaped. In this study, we used bluetongue virus (BTV) and its natural sheep host, to characterise the early events of an arbovirus infection with particular focus on the LNs. Our findings reveal a previously uncharacterized mechanism used by an arbovirus to manipulate host immunity. This study shows that BTV, similarly to other antigens delivered through the skin, is transported rapidly via the lymph to the peripheral lymph nodes. Here, BTV infects and disrupts the stromal network of marginal reticular cells and follicular dendritic cells composing the scaffolding of the follicular area. These cells contribute to antigen presentation and affinity maturation of B-cells for the production of antibodies. Consequently, we observed a loss of germinal centre structure, which hinders B-cell proliferation. This process results in a delayed production of high affinity and virus neutralizing antibodies that is directly related to the virulence of the BTV strain used and the severity of disease. Moreover the humoral immune response to a different antigen is also hampered in BTV-infected animals. Our data show that an arbovirus can evade the host antiviral responses by inducing an acute immunosuppression. Although transient, this immunosuppression occurs at the critical early stages of infection when a delayed host humoral immune response likely affects virus systemic dissemination and the clinical outcome of disease.

The objective. of this project was to determine the protein specificity of the cytotoxic T- lymphocyte (CTL) response to rotavirus infection in mice and to assess the rotavirus serotype/strain independent nature of this response. Previous work, involving the rotavirus group at Warwick, had shown that the outer shell glycoprotein VP7 is a major target antigen for a CTL response that is virus serotype-independent. However, that work did not cover all twelve rotavirus proteins, was confined to one strain of adult mice (C57BL/6, H- 2b) and covered only two of the fourteen VP7 serotypes (serotypes 3 and 6) (Offit et al., 1994). Recombinant vaccinia viruses expressing individual rotavirus UKtc proteins VP2, VP3, NS26 and NS12 were constructed to complete a set of recombinant vaccinia viruses covering the full complement of rotavirus proteins from the bovine UKtc strain. These were used to define rotavirus proteins eliciting a CTL response in three different mouse haplotypes. UKtc NS53 and UKtc VP7 stimulated a strong CTL response only in the H-2b MHC class I haplotype (UKtc NS53 and UKtc VP7 were restricted at H-2Db and H-2K b, respectively). Conversely, UKtc VP3 stimulated a strong CTL response in the H-2d and H-2k (but not in H-2b) MHC class I haplotypes. Work using congenic mouse strains was used to verify that the VP7 protein specific CTL response is restricted solely by the MHC class I antigens. Rotavirus RRV NS53 was not only found to elicit a CTL response in the H-2b MHC class I haplotype, similar to UKtc NS53, but also in the H-2d MHC class I haplotype. Thus, the individual rotavirus protein that elicits a CTL response not only depends on the MHC class I haplotype, but also on the actual rotavirus strain being tested. Many of the previous studies looking at the CTL response-to individual rotavirus proteins have, unlike this study, used several different rotavirus strains and, therefore, may have given an inaccurate representation of the rotavirus proteins that elicit a CTL response. Recombinant vaccinia viruses were also used to examine the serotype/strain independent nature of the CTL response against the major target antigens. The analysis was extended to cover VP3 from two different strains, NS53 from three different strains and VP7 from seven of the fourteen serotypes. VP3 and NS53 were found to elicit a strain- dependent response whereas the serotype-independent nature of the CTL response to VP7 was confirmed. Since the serotype-independent nature of the rotavirus VP7-specific CTL response was found to cross-protect between half of the VP7 serotypes, irrespective of the immunising. serotype, it would be reasonable to speculate that the CTL response is serotype-independent between all the VP7 serotypes. Finally, recombinant vaccinia viruses were used to locate CTL epitopes on NS53 and VP7. Recombinant vaccinia virus expressing a UKtc NS53 deletant mutant (P9DM5) showed there to be at least one strain specific epitope in the first 150 amino acids of the UKtc NS53 protein. Recombinant vaccinia viruses expressing four different UKtc VP7 fragments spanning 46% of this protein were examined. It was found that the fragment spanning the restriction enzyme sites at nucleotide 90 (CIaI) and nucleotide 196 (Hhal), i. e. between amino acids 13 and 48 of the mature UKtc VP7 protein, contained a serotype- dependent CTL epitope. The finding suggests that the immunodominant epitope identified in the same region of VP7 by Franco et al. (1993) was not the serotype-independent epitope.

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Background: There are a large number of viruses spread by mosquitoes, many of which cause debilitating, often fatal, neurological disease such as acute encephalitis. In this study we have used two different neurotropic viruses: Semliki Forest virus (SFV), and West Nile virus (WNV), both of which can cause severe panencephalitis in the mouse. The influx of leukocytes into the infected tissues is mediated by chemokines and is believed to be important for virus clearance. To date, we have only limited insights into the precise nature of chemokine involvement, and an improved understanding of these important axes provides a new target for the development of novel therapies. Hypothesis: Based on previous studies investigating the role of chemokines during neuroinflammation it was hypothesised that chemokines and other cytokines are highly upregulated during viral encephalitis, and the blockade of selected chemokine receptors would lead to altered disease outcome. It was also hypothesised that chemokine receptors would present plausible targets for the treatment of viral encephalitis. Results: To test these hypotheses, the chemokine expression pattern and the kinetics of chemokine mediated leukocyte recruitment during viral encephalitis were analysed in unprecedented detail by TaqMan low density array, and flow cytometry, respectively, and key chemokine receptor were identified as therapeutic targets. Both SFV and WNV exhibited a similar pattern of chemokine upregulation, although WNV induced significantly higher fold expression. The key chemokines upregulated were CCL2, 3, 5, 7, CXCL9 and CXCL10. The upregulation of chemokines coincided with leukocyte influx into the CNS. After identifying the key chemokines upregulated during viral encephalitis, next a selected panel of chemokine receptor antagonists was utilized to evaluate the hierarchy and relative importance of distinct chemokine receptors for CNS leukocyte influx, viral clearance, neuropathogenesis and host survival. We identified the CXCR3 axis as being the key instigator of CNS inflammation in response to alphavirus infection, placing it at the top of a hierarchal cascade that is followed by CCR2 and CCR5. Critically, inhibition of both CXCR3 and CCR2 simultaneously, significantly improved host survival to otherwise lethal encephalitis. Conclusion: These data suggest that chemokine receptors represent plausible therapeutic targets for viral encephalitis.

Coinfection with bacteria and viruses is an understudied area of microbiology, despite its potential to modulate pathogen abundance and host survival. We investigated the effect of bacteria on virus infection and developed an in vitro system to study the first step: viral internalization. Our studies show that multiple bacterial species promote the entry of a diverse panel of viruses into lung and gut epithelial cells. Bacteria expressing the toll-like receptor (TLR)5 agonist, flagellin, are most efficient at inducing viral uptake and studies using recombinant flagellin or aflagellate bacterial strains confirm that flagellin has pro-viral activity. Flagellin promotes epithelial cells to support virus entry via TLR5-dependent activation of NF-KB. To extend these observations and study the role of flagellin in the complete viral replicative lifecycle, we studied human immunodeficiency virus (HIV)-1 replication in T cells. Flagellin augments HIV-1 entry and promoter activity and increases the production of extracellular virus. The data presented in this thesis highlight a new role for bacterial flagellin to promote diverse virus infection of epithelial barriers and enhance the spread of HIV-1. This has significant implications for understanding how exposure to multiple pathogens can alter susceptibility to infection and its associated pathogenesis.

Outer membrane vesicles (OMVs) are nanosized proteoliposomes ubiquitously released from the outer membrane of Gram negative bacteria, and are known to contribute to immune priming and disease pathogenesis. However, the current understanding of their interactions with host cells is limited by a lack of methods to study the rapid kinetics of vesicle entry and cargo delivery. This work has developed a highly sensitive method to study vesicle entry into host cells in real-time using a genetically encoded, vesicle targeted probe. Using this approach, it was found that the route of vesicular uptake, and thus entry kinetics and efficiency, are shaped by bacterial cell wall composition. The presence of O polysaccharide in lipopolysaccharide creates a bias towards non-receptor mediated endocytosis, which enhances both the rate and efficiency of entry into host cells. This work indicates that the composition of the bacterial cell wall influences the behaviour of OMVs, and is therefore implicated in secretion-system independent delivery of bacterial virulence factors during Gram negative infection.